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WO2009100449A1 - Procédés de dosage de réseau dynamique - Google Patents

Procédés de dosage de réseau dynamique Download PDF

Info

Publication number
WO2009100449A1
WO2009100449A1 PCT/US2009/033586 US2009033586W WO2009100449A1 WO 2009100449 A1 WO2009100449 A1 WO 2009100449A1 US 2009033586 W US2009033586 W US 2009033586W WO 2009100449 A1 WO2009100449 A1 WO 2009100449A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
probe
pcr
primers
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2009/033586
Other languages
English (en)
Inventor
Kenneth J. Livak
Marc Unger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Standard Biotools Inc
Original Assignee
Fluidigm Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fluidigm Corp filed Critical Fluidigm Corp
Priority to US12/866,018 priority Critical patent/US9157116B2/en
Publication of WO2009100449A1 publication Critical patent/WO2009100449A1/fr
Anticipated expiration legal-status Critical
Priority to US14/880,112 priority patent/US20160153026A1/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • a metno ⁇ ior me ⁇ etection oi a plurality oi nucleic acid analytes may combining aliquots of nucleic acid containing sample with labeled nucleic acid probes and PCR primers in separate compartments of a microfluidic device and segregating the aliquots from each other, performing a homogeneous assay, and querying each of the samples for the presence of a target nucleic acid analyte.
  • the nucleic acid containing sample may be a cDNA containing sample.
  • the assay method may further include performing a preliminary amplification reaction on the nucleic acid sample to generate a pre-amplified sample.
  • the preliminary amplification reaction may include making a reaction mix containing the nucleic acid containing sample, the forward primers, and the reverse primers, and then subjecting the reaction mix to PCR amplification.
  • the reaction mix may be subjected in a range of about 10 cycles to about 18 cycles of PCR amplication. In particular, the reaction mix may be subjected to about 14 cycles of PCR amplification.
  • FIGURE 4 is a schematic illustrating the combinational mixing of forward and reverse primers in order to generate functional PCR assays on a 4 x 4 device according to principles of the invention.
  • FIGURE 5 is a schematic illustrating the 4 x 4 device of Figure 1 after the rows and columns have been filled according to principles of the invention.
  • LNA is locked nucleic acid
  • adjacent generally refers to the positioning of the primer with respect to the probe on its complementary strand of the target nucleic acid analyte.
  • the primer and probe may be separated in a range of about 1 to about 20 nucleotides, more specifically, in a range of about 1 to about 10 nucleotides, or may directly abut one another.
  • label refers to any atom or molecule which can be used to provide a detectable and/or quantifiable signal.
  • the label can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetric, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
  • nucleic acid generally refers to cDNA, DNA, RNA, single-stranded or double-stranded and any chemical modification thereof, such as PNA and LNA. LNAs are described in U.S. Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748 herein incorporated by reference in their entirety. Nucleic acids may be of any size. Nucleic acid modifications may include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole.
  • Such modifications may include modified bases such as 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocylcic amines, substitutions of 5-bromo-uracil, backbone modifications, methylations, unusual base pairing combinations such as the isobases isocytidine and isoguanidine and the like.
  • the nucleic acid can be derived from a completely chemical synthesis process, such as a solid phase mediated chemical synthesis, or from a biological origin, such as through isolation from almost any species that can provide nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes.
  • PLP padlock probe
  • the sequences at the 3' and 5' ends of the PLP are complementary to adjacent sequences in me iargei nucieic aci ⁇ anaiyre.
  • m me central, noncomplementary region of the PLP there is a "tag sequence” that may be used to identify the specific PLP.
  • the tag sequence may be flanked by universal primer sites or unique and/or specific primer sites, which allow PCR amplification of the tag sequence.
  • the 5' and 3' ends of the PLP are brought into close proximity and may be subsequently ligated.
  • the resulting product is a circular probe molecule catenated to the target nucleic acid analyte.
  • the tag regions of circularized PLPs may be amplified and quantified and/or detected using TAQMAN® Real Time PCR, for example.
  • the presence and amount of amplicon may be correlated with the presence and quantity of target sequence in the sample.
  • PLPs see, e.g., Landegren et al., 2003, Padlock and proximity probes for in situ and array-based analyses: tools for the post-genomic era, Comparative and Functional Genomics 4:525-30; Nilsson et al., 2006, Analyzing genes using closing and replicating circles Trends Biotechnol. 24:83-8; Nilsson et al., 1994, Padlock probes: circularizing oligonucleotides for localized DNA detection, Science 265:2085-8.
  • the above references are incorporated by reference herein in their entirety.
  • the oligonucleotide primer typically contains in the range of about 15 to about 30 nucleotides, although it may contain more or fewer nucleotides.
  • the primers should be sufficiently complementary to selectively anneal to their respective strands and form stable duplexes.
  • One skilled in the art appreciates how to select appropriate PCR primer pairs to amplify the target nucleic acid analyte of interest.
  • Aliquots of the sample and forward primers are distributed into separated compartments of a microfluidic device and combined with the appropriate reagents.
  • the aliquot may have a volume of in the range of about 1 picoliter to about 500 nanoliters, more often in the range of about 100 picoliters to about 20 nanoliters, even more often in the range of about 1 nanoliter to about 20 nanoliters, and most often in the range of about 5 nanoliters to about 15 nanoliters.
  • the reagents may include a labeled nucleic acid probe, PCR primers (e.g., forward primers and reverse primers), a thermostable DNA polymerase, an aqueous buffer, magnesium chloride and deoxynucleotide triphosphates, and may also include other non-reactive ingredients.
  • the sample may be pre-amplified prior to pooling with the forward primers.
  • the reverse transcribed sample is mixed with all forward and reverse primers and then subjected to about 10 cycles to about 18 cycles of PCR. Using 14 PCR cycles, the pre-amplification reaction increases the nucleic acid analytes by about 16,000 fold.
  • the pre-amplification reaction is described in more detail in Specific Example 1 , below.
  • the homogenous assay of the invention should not be construed to be limited to PCR-based detection methods, but may employ any method of detection and /or quantification to detect and/or quantify a target nucleic acid analyte.
  • PCR may be used to amplify a target.
  • other amplification systems or detection systems may be used, including systems described in U.S. Pat. No. 7,118,910, which is incorporated herein by reference in its entirety.
  • a detection system other than PCR may be used such as an Invader® assay (Third Wave, Madison, WI).
  • the thermal cycling protocol consisted of 50 0 C for 2 min, 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C, 5 sec at 70 0 C, and 1 min at 6O 0 C.
  • the final concentrations in the reaction chambers of the array were 200 nM each forward and reverse primers and 100 nM UPL probe.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés de haute capacité utilisés pour combiner les particularités de l’utilisation d’un dispositif microfluidique de type matrice, des sondes d’acides nucléiques marquées et des dosages homogènes pour détecter et/ou quantifier des analytes d’acides nucléiques. Des procédés de haute capacité sont capables de détecter des analytes d’acides nucléiques avec une haute spécificité PCR et de sonde, produisant un faible fond de fluorescence et donc un rapport signal sur bruit élevé. De plus, des procédés de haute capacité sont capables de détecter un analyte d’acides nucléiques en faible nombre de copie par cellule.
PCT/US2009/033586 2008-02-08 2009-02-09 Procédés de dosage de réseau dynamique Ceased WO2009100449A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US12/866,018 US9157116B2 (en) 2008-02-08 2009-02-09 Combinatorial amplification and detection of nucleic acids
US14/880,112 US20160153026A1 (en) 2008-02-08 2015-10-09 Dynamic Array Assay Methods

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2739008P 2008-02-08 2008-02-08
US61/027,390 2008-02-08

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US12/866,018 A-371-Of-International US9157116B2 (en) 2008-02-08 2009-02-09 Combinatorial amplification and detection of nucleic acids
US14/880,112 Continuation US20160153026A1 (en) 2008-02-08 2015-10-09 Dynamic Array Assay Methods

Publications (1)

Publication Number Publication Date
WO2009100449A1 true WO2009100449A1 (fr) 2009-08-13

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/033586 Ceased WO2009100449A1 (fr) 2008-02-08 2009-02-09 Procédés de dosage de réseau dynamique

Country Status (2)

Country Link
US (2) US9157116B2 (fr)
WO (1) WO2009100449A1 (fr)

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